The long term goal of this project is to understand, on the molecular level, the regulation of enzyme activity by covalent modification and allosteric phenomena. The two enzymes that will be investigated in the next five year period are E. coli glutamine synthetase and carbamoyl-phosphate synthetase. These enzymes are essential for nitrogen metabolism in procaryotes and eucaryotes and their catalytic activity is regulated by multiple mechanisms. The studies will focus on how structure and function are related in the catalytic mechanism as well as how each mode of regulation influences catalysis. The specific aims are: 1) To determine the individual rate constants in the mechanism of both glutamine synthetase and carbamoyl-P synthetase using rapid-quench, isotope partitioning and positional isotope exhange methods. 2) To study the influence of allosteric effector molecules on the mechanism of both enzymes. Glutamine synthetase is regulated by many (7 or more) feedback modifiers and we plan to evaluate how each effector alters individual rate constants using multiple kinetic methods. Carbamoyl-P synthetase is activated by IMP and ornithine and inhibited by UMP and the changes in substrate binding and individual rate constants elicited by these effectors will be determined. 3) To determine how covalent adenylylation of individual subunits of glutamine synthetase alters metal ion specificity and enzyme activity. Several kinetic techniques will be used to determine how substrate binding and/or individual rate steps are altered by this method of covalent regulation. 4) To construct a topographical map describing the three dimensional relationships among the active and allosteric sites of glutamine synthetase and carbamoyl-P synthetase. Nmr, epr and fluorescence energy transfer experiments will be used. 5) To determine which amino acids are involved in substrate binding and catalysis of glutamine synthetase. convalent modification of the ATP site by 5'-p-fluorosulfonylbenzoyladenosine was accomplished and the modified amino acid will be identified. Potential active site lysines have been modified by reductive methylation and their position in peptides will be evaluated. Kinetic studies of the pH-rate profiles of the substrates will continue to be aimed at implicating certain enzymatic groups in substrate binding and catalysis. 6) To determine the DNA sequence of the glnA gene that codes for glutamine synthetase in E. coli. This gene has been cloned in pBR322. 7) To use oligonucleotide direct mutagenesis to alter individual amino acids of glutamine synthetase that are essential for regulation and catalysis.